The general goal is to identify and understand the molecular mechanisms of recombinational DNA repair, a key pathway for preserving genomic integrity. This application focuses on the Rad51 paralogs, a class of proteins that has been notoriously difficult to study, and whose mechanistic contribution to recombination remains to be defined. The Rad51 paralogs act at a pivotal point, namely the assembly and maintenance of the Rad51- ssDNA filament, an essential intermediate for high-fidelity template-directed repair by homologous recombination DNA. In this application, two principal investigators (Heyer and Kowalczykowski) combine their expertise in a comprehensive approach to advance our understanding of Rad51 paralog function in DNA repair in yeast and humans utilizing single-molecule biophysics, ensemble biochemistry, and yeast genetics. The Specific Aims are: (1) Determine the functional specialization of yeast Rad51 paralogs in yRad51-ssDNA filament assembly. The presynaptic Rad51-ssDNA filament exists in a meta-stable balance between its assembly and disassembly. We will determine the functional specialization of the Rad51 paralogs in Rad51 filament formation at gaps versus DSB breaks and determine potential selectivity for specific anti-recombinases. (2) Determine functional consequences of human RAD51 paralog action. To ascertain the functions of the RAD51 paralogs, their biochemical behavior will be examined. We will determine their DNA binding specificity, their capacity to stimulate DNA strand exchange promoted by hRAD51, and their ability to stabilize hRAD51 nucleoprotein filaments from disassembly both intrinsically and by helicases (BLM, RECQL5, FANCJ, and FBH1); the mechanisms underlying any effect will be determined. We will also examine whether other proteins, which interact with the RAD51 paralogs, function to regulate the formation or stability of RAD51 filaments. (3) Use single-molecule analyses to determine how the human RAD51 paralogs affect the dynamic behavior of hRAD51-ssDNA filaments. Testing the paradigm established in the yeast studies, we will measure whether the human RAD51 paralogs alter the rate of hRAD51 filament assembly or disassembly. The paralogs could, in principle, increase the nucleation frequency or the filament growth rate, or they could decrease the dissociation rate. These measurements will use direct imaging by single-molecule fluorescence microscopy of hRAD51 filament dynamics on individual molecules of DNA.